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Genome plasticity in soil bacteria is predicted to be evolutionarily advantageous, allowing bacteria to sample genetic variation for adaptation to local soil ecology. In the field population of mesorhizobia where the symbiosis island (ICEMlSym[R7A]; an I̲ntegrative C̲onjugative E̲lement) was first identified, individual members were found to have significant chromosomal variation downstream of the phe-tRNA gene or phe-tRNA integrated ICEMlSym[R7A]. However, the nature of this genetic variation and whether it contributed to the adaptation of the indigenous mesorhizobia to their field environment were unknown. This work focused on a nodule isolate, Mesorhizobium sp. strain R88B, a member of the indigenous mesorhizobial population that received ICEMlSym[R7A] from strain R7A. The region downstream of ICEMlSym[R7A] was sequenced, revealing three distinct regions of non-conserved DNA, totalling 34.5 kb. Integrated directly downstream of ICEMlSym[R7A] was IMEMlAdh[R88B], a 24.3-kb novel I̲ntegrative M̲obilisable E̲lement. Using a PCR-based assay, it was shown that the IMEMlAdh[R88B] integrase could excise not only IMEMlAdh[R88B], but also a dual-IMEMlAdh[R88B]/ICEMlSym[R7A] hybrid, indicating the potential mobility of IMEMlAdh[R88B], and a likely evolutionary intermediate of a novel ICE. However, a functional role for MadA, (a putative adhesin and the sole adaptive trait encoded on IMEMlAdh[R88B]) was not discovered. Southern hybridisations with the mesorhizobial population provided evidence for the existence of a novel family of IMEs in the mesorhizobia, which, by diversifying their internal sequences, provide allele-specific variation to the population. The two other regions downstream of IMEMlAdh[R88B] possessed no obvious mobile genetic element structures, and only the region adjacent to the core-chromosome encoded ORFs with putative functions. Mutation of two of these ORFs, fhuD1 and fhuB1, identified their function as two of the four components of a ferrichrome ABC-uptake (Fhu) system. Using genetic screens, the remaining components of this transporter were mapped to two separate loci. Thus, the functional transporter in R88B was a composite of at least two independently-acquired Fhu systems. The genetic screens also revealed that ferrichrome utilisation was dependent on a TonB energy-transduction system encoded downstream of the Fhu ATPase gene, fhuC. Expression studies on the three fhu loci demonstrated that, despite their separate acquisition, their expression was coordinately up-regulated in response to low-iron conditions. Bioinformatics on the predicted promoter regions of the fhu genes identified the binding site of the rhizobial Fur analogue, RirA, which is likely to be responsible for this expression profile. Southern hybridisations of DNA isolated from members of the mesorhizobial population revealed the three fhu loci were not conserved in the mesorhizobial population. The presence of FhuA was the best predictive marker for the trait. It is proposed that multiple rounds of acquisitions and recombinations, both illegitimate and legitimate, formed this transporter, with the constant need for iron offset by the negative selection pressure of FhuA being a target for phage. None of the Fhu-specific genes was present in the sequenced M. loti strain MAFF303099 though flanking sequences were, further emphasizing the role of genome microevolution in forming the Fhu phenotype.

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.

Although legumes have been widely studied for their nitrogen-fixing ability, it is uncertain to what extent legume cover crops achieve their nitrogen-fixing potential under the climatic conditions encountered in western Oregon. Furthermore, it is unknown what factors control the proportions of legume cover crop N that are either sequestered into soil organic matter, or that contribute to the N requirements of the following summer crop. Soil was sampled in mid-September 1997, after harvest of a summer broccoli crop, from plots located at the North Willamette Research and Extension Center, Aurora, Oregon. Soil was sampled from main plots that had been either winter cover cropped with red clover (LN��� and LN���) or fallowed during the winter period (FN��� and FN���), and specifically from sub-plots in which the following summer crop had received either zero (N���) or an intermediate (N���) rate of N fertilizer as urea. Levels of total organic carbon (TOC), total Kjeldahl nitrogen (TKN), and readily mineralizable C and N were measured in both whole soil samples and in different aggregate-size classes (<0.25, 0.25-0.5 0.5-1.0, 1.0-2.0, and 2-5mm) prepared by dry sieving the soil. Aggregate size-class distribution was not affected by the cover crop treatment. Although there was no significant effect of cover crop treatment on either TKN or TOC levels in whole soil samples, TOC levels were consistently higher in the small aggregate size-classes <1 mm of the fallow than the legume treatment. There was a significantly higher level of mineralizable C in the <0.25 mm size class of the legume than the fallow treatment. There was a trend for the level of mineralizable N to be greater in soil from the legume than the fallow treatment. However, N fertilizer had a significant positive effect on the level of readily mineralizable N in both fallow and legume cover-cropped treatments, it had a negative effect on TKN levels among all aggregate-size classes. There were differences in the levels of mineralizable N measured among the aggregate-size classes, and immobilization of N between 20 and 40 days of incubation also differed among the aggregate-size classes. / Graduation date: 1999

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 6, 2010. Major professor: Tryg Lundquist, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Civil and Environmental Engineering." "November 2009." Includes bibliographical references (p. 43-47).

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 222-247). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 222-247). Also available on the Internet.

In the N2 fixing legume symbiosis, mineral nitrogen (N) not only decreases N2 fixation, but also delays and inhibits the formation and development of nodules. The purposes of this thesis were to elucidate the role of signaling in the mineral N effects on nodulation and nitrogen fixation in soybean [Glycine max (L.) Merr.] and to attempt to find ways to overcome this inhibition. The responses of soybean plants, in terms of daidzein and genistein synthesis and exudation, to different mineral N levels were studied. Daidzein and genistein distribution patterns varied with plant organs, mineral N levels, and plant development stages. Mineral N inhibited daidzein and genistein contents and concentrations in soybean root and shoot extracts, but did not affect root daidzein and genistein excretion in the same way. In both synthesis and excretion, daidzein and genistein were not affected equally by mineral N treatments. Variability existed among soybean cultivars in the responses of root daidzein and genistein contents and concentrations to mineral N levels. The amount of daidzein and genistein excreted by soybean roots did not always correspond to the daidzein and genistein contents and concentrations inside the roots. On the Bradyrhizobium japonicum side, nod gene expression was inhibited by mineral nitrogen. Genistein was used to pre-incubate B. japonicum cells or was applied directly into the plant growing medium. The results showed that genistein manipulation increased nodule weight and nodule nitrogen fixation under greenhouse conditions, but interactions existed among soybean cultivars, genistein concentrations and nitrate levels. Similar results were found under field conditions. Soybean yield was increased on sandy-loam soil by preincubation of B. japonicum cells with genistein. Addition of genistein beginning at the onset of nitrogen fixation also improved soybean nodulation and nitrogen fixation. Soybean cultivars had different sensitivities to genistein additi / Other studies also show that temperature affected genistein and daidzein content and concentration in soybean roots. The effect of temperature varied among soybean cultivars. Some PGPR strains can mitigate the negative effects of nitrate on soybean nodulation and nitrogen fixation, however, this is influenced by soybean genotype. Applying PGPR together with genistein preincubation of B. japonicum cells improved soybean nodulation and increased yield. The level of improvement varied among soybean cultivars and PGPR strains. Preincubation of B. japonicum cells with genistein improved strain competitiveness under greenhouse, but not field conditions. / Overall, these findings suggested that both plant-to-Bradyrhizobium and Bradyrhizobium-to-plant signals play important roles in the effects of mineral N on nodulation and N fixation. Signal manipulation could partially overcome the inhibitory effects of mineral N on soybean- B. japonicum N fixation symbiosis.

Nitrogen (N) generally limits primary production across large areas of the world's oceans. Allochthonous inputs of N (i.e., "new" N) via N2-fixing organisms (diazotrophs) are crucial for sustaining primary production and are often associated with net export of organic matter (OM) from surface waters. Diazotroph N (ND) contribution plays an integral role in supporting oceanic food webs and regulating the flux of OM into and through the oceans (e.g., the biological pump). Stable isotope techniques were used to trace the input and movement of new N through oceanic food webs. Laboratory experiments were performed to determine elemental and isotopic shifts of OM exposed to microbial and metazoan processing. δ15N of OM was typically higher when exposed to microbial communities, with no difference in δ15N of OM between experiments incubated at different temperatures (4°C and 25°C). In separate experiments, shrimp digestion did not alter the δ15N of OM through digestion, but the δ15N of macerated OM was enriched in 15N. Both of these experiments provide insight into the mechanisms driving variations in the δ15N of OM in the world's oceans. To assess the role of diazotrophs in oceanic food webs, we used the distribution of δ15N to quantify the relative ND contribution to suspended particle N (PN) and mesozooplankton N biomass (NZOOP) in the subtropical North Atlantic (STNA). Qualitatively, ND contribution was often high for both PN and NZOOP, with the highest contributions occurring in the mixed layer. Our results also indicate higher ND contribution to both PN and NZOOP in the western portion of the basin than in the east. ND contribution to larger mesozooplankton at depth further suggests that migrating mesozooplankton transport ND out of the mixed layer. Quantitatively, ND trophic transfer efficiency was lower than bulk N trophic transfer efficiency, suggesting low assimilation of ND by mesozooplankton. Overall, we estimated a ND pool turnover time on the order of weeks for our region of study. These findings demonstrate that ND is laterally and vertically variable in the STNA, and that the ND pool is sensitive to perturbations on short timescales. We discuss the global implications of our findings and their implications for the N cycle and elemental fluxes through oligotrophic oceans.

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.